Since Szentivanyi proposed in 1968 that asthma might be due to an inherited or acquired deficit in beta-adrenergic function, much research has focused on the beta adrenergic receptor (beta2AR) and the beta-adrenergic cascade. However, 22 years later the question is still unsettled, as research has focused largely on the leukocyte, which is a poor model of human asthma. Studies of bronchial muscle tissue from animal models of asthma and from human asthmatics obtained either at surgery or at autopsy show a very definite deficit in beta-adrenergic function. However, the intracellular events underlying this beta adrenergic deficit is unknown. We have a well characterized animal model of airway hyperresponsiveness that clearly demonstrates decreased sensitivity to beta adrenergic agonists in vivo and in vitro. We have access to human asthmatic airway tissue which shows a similar defect, and we have access to a population of well characterized human asthmatics. We hypothesize that the decreased sensitivity to beta adrenergic agonists relates to a specific defect either in generation or degradation of intracellular cAMP. To identify directly the intracellular lesion we plan to measure numbers and affinities of beta2AR and muscarinic receptor subtypes, second messengers, adenylyl cyclase and phospholipase C and their regulation by the signal transducing G proteins in the airway smooth muscle of the basenji-greyhound dog, and to understand the mechanism by which glucocorticoids modify the response. We will apply the insights gained in the animal model to similar studies with available human airway tissues. In addition, we will determine whether genetic linkage exists between inheritance of the asthma phenotype and specific beta2AR gene alleles or other genes (e.g. G proteins) involved in beta adrenergic functions, and characterize specific mutations by nucleotide sequencing. The reduced in vitro sensitivity to beta agonists in our animal model provides us with the unique opportunity of easy accessibility to large quantities of airway smooth muscle for our in vitro studies from two allergic lines of animals - one hyperresponsive and one normoresponsive, allowing us to focus on the deficit leading to hyperresponsiveness, not confounded by allergy or drug therapy. We also have the unique ability to control and manipulate environmental and genetic influences. Results from the proposed work will provide new information about the intracellular mechanisms which regulate airway smooth muscle tone in this model. We will apply this information to the limited tissues available in humans and to human asthmatics using a candidate gene approach. We think this three pronged approach, using leukocyte DNA, human airway tissue and tissue from the BG dog model of airway hyperresponsiveness will yield important new insights. Since glucocorticoids and beta agonists remain the mainstay of asthma treatment, it is critical to elucidate the molecular mechanism of reduced beta agonist sensitivity and its modulation by glucocorticoids in patients and animal models of asthma.